RSC

Chemical Biology
View Article Online
PAPER View Journal | View Issue

Covalently attached intercalators restore duplex

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

stability and splice-switching activity to triazole-

Cite this: RSC Chem. Biol., 2022,
modified oligonucleotides†
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

3, 765

Anna Dysko,a Ysobel R. Baker, a Graham McClorey,b Matthew J. A. Wood,b

Sabine Fenner,c Glynn Williams,‡c Afaf El-Sagheerad and Tom Brown *a

Oligonucleotides are rapidly emerging as powerful therapeutics for hard to treat diseases. Short single-
stranded oligonucleotides can base pair with target RNA and alter gene expression, providing an
attractive therapeutic approach at the genetic level. Whilst conceptually appealing, oligonucleotides
require chemical modification for clinical use. One emerging approach is to substitute the
phosphodiester backbone with other chemical linkages such as triazole. The triazole linkage is inherently
Received 13th April 2022, resistant to enzymatic degradation, providing stability in vivo, and is uncharged, potentially improving
Accepted 15th May 2022 cell-penetration and in vivo distribution. Triazole linkages, however, are known to reduce RNA target
DOI: 10.1039/d2cb00100d binding affinity. Here we show that by attaching pyrene or anthraquinone to the ribose sugar on the 5 0 -
side of the triazole, it is possible to recover duplex stability and restore the splice switching ability of
[Link]/rsc-chembio triazole-containing oligonucleotides.

Introduction Without chemical modification, oligonucleotides (ONs) are

rapidly degraded by nucleases in biological environments
Antisense oligonucleotides (ASOs) are rapidly emerging as rendering them ineffective as therapeutics. For this reason,
powerful agents for treating genetic diseases.1–3 These short chemical modifications have been developed to provide protec-
single stranded analogues of DNA (typically 15–25 bases in tion against nucleases and enhance pharmacokinetic
length) can bind to their complementary RNA targets and alter properties.10 Commonly used chemical modifications for ASOs
gene expression by a variety of mechanisms.4 ASOs can inhibit include replacing phosphodiester (PO) linkages in the back-
translation of specific proteins by recruiting ribonuclease H1 bone with phosphorothioate groups (PS)11,12 to improve duplex
(RNase H) to degrade the target messenger RNA (mRNA),5 or by stability against enzymatic degradation and cellular uptake,
binding to mRNA translational start sites, thereby blocking the and ribose modifications to improve target affinity and lipo-
binding of ribosomes.6 ASOs can also be designed to bind philicity. These include 2 0 -F, 2 0 -OMe, 2 0 -O-(2-methoxyethyl) and
precursor mRNA (pre-mRNA) and modulate RNA splicing.7–9 locked nucleic acids (LNAs).11,13–15 These modifications have
This can introduce an out-of-frame deletion, triggering non- transformed the field, and the FDA has now approved several
sense mediated decay of the transcript. Alternatively, it can ASOs for clinical use.16 However, the widespread therapeutic
correct aberrant splicing caused by splice point mutations, or applications of ASOs are still hampered by potential toxicity
truncate the mRNA by removing exons that contain premature and their inability to efficiently cross cellular membranes.
stop codons or out-of-frame deletion mutations. Hence, there remains a need for new ASO chemistries to reduce
dosage, toxicity, cost and improve clinical efficacy.
Charge-neutral backbone modifications offer an intriguing
a
Department of Chemistry, Chemistry Research Laboratory, University of Oxford,
alternative for ASO development.17–19 Here, the PO backbone is
12 Mansfield Road, Oxford, OX1 3TA, UK. E-mail: [Link]@[Link]
b
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford,
partially or fully replaced with linkages that have no charge
UK (each phosphodiester linkage carries an anionic charge,
c
GSK Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, Fig. 1A). These linkages can confer nuclease resistance
SG1 2NY, UK (in vivo stability) and alter the overall anionic nature and
d
Chemistry Branch Department of Science and Mathematics, Faculty of Petroleum
lipophilicity of an ASO, potentially aiding cellular uptake and
and Mining Engineering, Suez University, Suez 43721, Egypt
† Electronic supplementary information (ESI) available: Experimental methods
modulating protein interactions that influence in vivo distribu-
and supplementary data. See DOI: [Link] tion. The triazole linkage (Fig. 1B) has several characteristics
‡ Current address: Evotec, Abingdon, UK. that are desirable when developing new ASO candidates. It is

© 2022 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2022, 3, 765–772 | 765
 View Article Online

Paper RSC Chemical Biology

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

Fig. 2 (Top) Overview of ON synthesis strategy via amino-modified ONs.

(Bottom) reagents used to functionalise the ONs. Reagents and condi-
tions: (a) solid phase ON synthesis, (b) N-hydroxysuccinimide (NHS)
Fig. 1 Comparison of the PO backbone (A) and the biocompatible tria- labelling with 3, (c) nitrophenyl active ester (oNp) labelling with 4,
zole linkage (B). Current approaches to stabilise the linkage include using a (d) guanidine addition with 5. DMT = 4,4 0 -dimethoxytrityl.
G clamp (C) and replacing the ribose with a morpholino (D) or an LNA
sugar (E and F), dashed bonds are used to indicate different combinations
of LNA on the 5 0 or 3 0 were investigated.
that one phosphoramidite monomer can be used to screen
multiple modifications without requiring multistep synthesis
biocompatible and completely stable to endogenous nucleases,
of a new phosphoramidite for each modification of interest. In
it is simple to synthesise and is charge neutral.20,21 Despite
this study, three different moieties were investigated: pyrene
these advantages, the triazole linkage reduces the affinity of an
(PY) and anthraquinone (AQ) which are known to increase
ON for target RNA, and a single triazole incorporation can
duplex stability by intercalation and/or minor grove
significantly reduce the stability of ON : RNA duplexes.21–23
binding,30–37 and guanidine (Gu) which stabilises duplexes by
Several different approaches to overcome this have been
masking the charge repulsion between opposite nucleic acid
reported including the use of an aminoethoxyphenoxazine
strands, and potentially improves ON cellular uptake.38–40
nucleobase analogue (G-clamp) which increases duplex stability
Although aromatic groups such as pyrene or anthraquinone
by a combination of increased base stacking and an extra
stabilise unmodified duplexes due to intercalation or groove
hydrogen bond with guanine (Fig. 1C),23 and substitution of
binding, in this study, we specifically wanted to determine if
the flanking ribose sugars with a morpholino analogue
they could increase duplex stability when placed directly next to
(Fig. 1D),24 or an LNA derivative (Fig. 1E and F).21,25,26 Whilst
an artificial backbone. We also wanted to determine if cationic
several triazole linkage designs exist,22,27–29 this study focuses
guanidine stabilises duplexes when placed adjacent to an
on the structure shown in Fig. 1B due to its ease of synthesis. In
uncharged artificial backbone.
the current study, by incorporating various chemical moieties
The synthesis of phosphoramidites 1 and 2 started with propar-
adjacent to the triazole linkage that are known to stabilise ON
gylation of the 20 -hydroxy group in ribonucleoside 641 (Scheme 1).
duplexes, we have significantly improved the RNA binding
Staudinger reduction of the azide in 7 followed by protection of the
affinity of ONs.
amine in 8 using Fmoc-succinimide (Fmoc-OSu) gave nucleoside 9.
This was then joined to 50 -azidothymidine 1042 or benzoyl protected
Results and discussion 50 -azidodeoxycytidine 1143 using the CuI-catalysed alkyne-azide
cycloaddition (CuAAC) reaction44 to give the T–T dinucleoside 12
Synthesis of triazole modified ONs and the T–C dinucleoside 13 respectively. These were then phos-
We envisaged the development of universal phosphoramidite phitylated to give the phosphoramidite building blocks 1 and 2.
building blocks that would enable multiple incorporations of We then evaluated the labelling strategy shown in Fig. 2.
the triazole and allow the post-synthetic attachment of various ON1 (Table 1) was synthesised using phosphoramidite 1 and
chemical functionalities to the 2 0 -position of the ribose on the further functionalised. Commercially available 1-pyrenebutyric
5 0 -side of the triazole. The phosphoramidites (Fig. 2) were acid N-hydroxysuccinimide (NHS) ester 3 was used to attach
designed to be compatible with standard solid-phase ON pyrene with a butyryl linker to yield ON2. ON1 was converted to
synthesis and contain an amino group that can be functiona- ON3 using the active carbonate derivative of AQ 4 which
lised after ON synthesis. A key advantage to this approach is was prepared from the previously reported alcohol45,46 using

766 | RSC Chem. Biol., 2022, 3, 765–772 © 2022 The Author(s). Published by the Royal Society of Chemistry
 View Article Online

RSC Chemical Biology Paper

Biophysical evaluation
Single addition. UV melting was used to determine the
melting temperatures (Tms) (duplex stabilities) of ONs 1–9 with
complementary DNA and RNA targets. The Tm values are given
in Table 1 and melting derivatives of the modified duplexes are
shown in Fig. 3.
The data show that this approach can be used to overcome
the loss in duplex stability associated with backbone
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

modifications47 such as the charge-neutral triazole. In agree-

ment with previous reports, the triazole is destabilising com-
pared with the phosphodiester linkage,21–23 a DTm of 8.9 1C
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

was observed in duplexes with DNA and a DTm of 7.4 1C in

duplexes with RNA (compare ON1 with ON9). This indicates
that the 2 0 -aminoethoxy (AE) modification alone does not
Scheme 1 Synthesis of phosphoramidites 1 and 2. (a) NaH, propargyl
bromide, THF, reflux, 14 h, 72% (b) Ph3P, H2O, THF, 45 1C, 2 h (c) Fmoc- significantly improve the duplex stability of triazole modified
OSu, CH2Cl2, pyridine, r.t., 2 h, 65% over 2 steps (d) sodium ascorbate, ONs. Attachment of PY and AQ resulted in DNA duplexes that
CuSO4, (tris-(benzyltriazolylmethyl)amine ligand (TBTA), DMF, H2O, r.t., were 4.1 and 4.2 1C more stable than the unmodified DNA :
4 h, T = 86%, C(Bz) not fully purified. (e) 2-cyanoethyl-N,N- DNA duplex (compare ON2 : DNA and ON3 : DNA with ON9 :
diisopropylchlorophosphoramidite, DIPEA, CH2Cl2, r.t., 2.5 h, T = 34%,
DNA). PY and AQ also regained the loss in thermal stability of
C(Bz) = 55% over 2 steps.
duplexes with a complementary RNA target, albeit to a lesser
degree. The addition of PY to the triazole resulted in a duplex
with RNA that was 0.7 1C less stable than the unmodified
Table 1 Sequences of ONs and their melting temperatures (Tm) when duplex (compare ON9 : RNA with ON2 : RNA) but 6.7 1C more
hybridised with complementary DNA and RNA stable than the triazole modified ON1 (compare ON2 : RNA with
ON1 : RNA). AQ improved the stability of the triazole modified
DNA target RNA target
ON Sequence 5 0 to 3 0 Tm (DTm) 1C Tm (DTm) 1C DNA : RNA duplex to a greater degree than PY, with an increase
in Tm of 2.6 1C compared to unmodified DNA (compare ON3 :
1 53.0 (8.9) 54.6 (7.4) RNA with ON9 : RNA) and an increase of 10 1C compared to the
2 66.0 (+4.1) 61.3 (0.7) triazole modified DNA (compare ON3 : RNA with ON1 : RNA). It
3 66.1 (+4.2) 64.6 (+2.6) is worth noting that the Gu modification had little effect on the
4 53.4 (8.5) 54.2 (7.8) stability of duplexes with and without a triazole linkage, and
5 59.0 (1.9) 60.5 (1.5) was therefore not considered further. Guanidine is cationic at
6 68.4 (+6.5) 64.5 (+2.6) neutral pH, and stabilises unmodified duplexes by masking
7 69.5 (+7.6) 69.0 (+7.0) charge repulsion between opposite nucleic acid strands. How-
8 61.5 (0.4) 62.8 (+0.8) ever, in the modified duplexes guanidine is close to an
9 61.9 62 uncharged triazole linkage. It is likely that stabilisation does
not occur because there is no ‘anion–anion’ charge repulsion to
DTm = change with respect to the unmodified duplex; X = 2 0 -
aminoethoxy thymidine (AE–T); = pyrene labelled AE–T; anthra- neutralise at triazole site.
quinone labelled AE–T; = guanidinylated AE–T; t = position of a These findings suggest that it is not the addition of the PY
triazole in place of the phosphodiester; = triazole dimer 1; Melting and AQ modifications alone, but synergy between the triazole
temperatures were determined at 4 mM duplex concentration in 10 mM
NaH2PO4/Na2HPO4 buffer with 200 mM NaCl at pH 7.2. Sequence of
DNA target = GCTGCAAGCGTCG; Sequence of RNA target =
GCUGCAAGCGUCG.

4-nitrophenyl chloroformate. ON1 was guanadinylated using

1H-pyrazole-1-carboxamidine hydrochloride to give ON4.
Details for the labelling conditions and synthesis of 4 are given
in the supporting information. A second set of ONs with a
phosphodiester instead of a triazole linkage (ON5-8, Table 1)
was prepared for use as controls in the biophysical studies. ON5
was synthesised using a 2 0 -aminoethoxy thymidine (AE–T)
Fig. 3 UV melting studies of the triazole-modified ONs against DNA (Left)
phosphoramidite41 and labelled with NHS ester 3, active carbo- and RNA (Right). The first derivatives of the melting curves are shown. AE =
nate 4 and carboxamidine 5, as described above, to give ONs 2 0 -aminoethoxy, P = pyrene, AQ = anthraquinone, G = guanidine. For
6–8 respectively. sequences see Table 1.

© 2022 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2022, 3, 765–772 | 767
 View Article Online

Paper RSC Chemical Biology

and the PY/AQ modifications that give rise to the observed

stabilisation. For example, looking at destabilisation resulting
from triazole (ON1 : DNA DTm = 8.9 1C) and the stabilising
effect of the PY without the triazole (ON6 : DNA DTm = +6.5 1C) it
might be expected that a combination of the two modifications
would produce a duplex with slightly lower stability than the
unmodified control duplex; however, a significant increase in
stability is observed (+4.1 1C). The triazole only destabilises a
pyrene modified ON by 2.4 1C (compare ON6 : DNA with
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

ON2 : DNA) which is much lower than the 8.9 1C difference

between triazole-modified ON1 : DNA and the control ON9 :
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

DNA. Similar results were observed for the AQ modification

against DNA. This effect was also observed for the modified
ONs with RNA but to a lesser degree.
The duplex stabilisation conferred by the triazole-pyrene or
triazole-anthraquinone oligonucleotides against DNA (Table 1)
is slightly greater than against RNA. However, in these exam-
ples the majority of the oligonucleotide backbone is
deoxyribose-phosphodiester (i.e., natural DNA), which is rele-
vant to a number of diagnostic applications including real-time
PCR. However, the application described below involves cell Fig. 4 CD spectra of the modified DNA : RNA duplexes (top) and modified
DNA : DNA duplexes (bottom). For sequences see Table 1. Melting tem-
studies, where the target is RNA, and for which stability to peratures were determined at 4 mM duplex concentration in 10 mM
nucleases is essential. For this reason, we used a 2 0 -OMe RNA- NaH2PO4/Na2HPO4 buffer with 200 mM NaCl at pH 7.2. Sequence of
phosphorothioate backbone which is known to favour binding DNA target = GCTGCAAGCGTCG; sequence of RNA target =
to RNA targets over DNA targets, so in vivo selectivity of the GCUGCAAGCGUCG.
triazole-modified oligonucleotides for RNA over DNA should
not be an issue.
Circular dichroism (CD) studies show that triazole com-
Table 2 Thermal melting data and sequences of the PS/2 0 -OMe ONs
bined with PY or AQ has greater effect on the structure of
used in assay 1. Melting temperatures against complementary RNA
DNA : DNA duplexes than the corresponding DNA : RNA
duplexes (Fig. 4) suggesting that the mode of interaction in ON ON sequence (5 0 to 3 0 ) Tm 1C
the duplexes with a DNA target could be different to that for
10 65.9
duplexes with an RNA target. This could explain the additional 11 55.5
stability observed when duplexed with DNA compared to RNA. 12 49.4
When hybridised with complementary RNA, ON1–ON3 show
13 71.1
similar CD spectra, and they overlap with the unmodified
14 60.5
ON9 : RNA spectra almost perfectly (Fig. 4A). These constructs
display characteristic A-type signals with strong maxima at 15 56.7
270 nm and minima at 240 nm. In contrast, whilst the CD 16 74.1
spectra for ON1-3 retain B-type features (maxima at 280 nm and 17 51.1
minima at 250 nm) variations in the shift and peak intensity 18 45.1
were observed compared to the unmodified duplex. These 19 70.2
observations suggest that the triazole perturbs the B form
X = AE–T; = pyrene labelled AE–T; anthraquinone labelled AE–T;
structure and that the AQ and PY modifications restore the B t = position of a triazole in place of the phosphorothioate linkage; =
form (as observed by the slight blue shift in the peak at triazole dimer 1, = triazole dimer 2. Melting temperatures were
determined at 2 mM duplex concentration in 10 mM NaH2PO4/Na2HPO4
280 nm). Further structural studies are required to determine
buffer with 200 mM NaCl at pH 7.2. Sequence of RNA target =
whether this is a result of intercalation or groove binding. UGUAACUGAGGUAAGAGG.
Multiple additions and different backbones. A combination
of 2 0 -OMe and phosphorothioate (PS) is often used to improve
the properties of ASOs in vivo. Hence, we evaluated the effect of NHS active ester 3 as described earlier to give ONs 13–15 or with
single and multiple triazole linkages on RNA binding affinity in AQ-o-nitrophenyl active carbonate 4 to give ONs 16–18.
a model ASO sequence with a 2 0 -OMe/PS backbone.48 The ONs The Tms of the ONs with a complementary RNA strand were
(Table 2) with 1, 3, or 4 incorporations of the triazole linkage determined by UV-melting (Table 2). Incorporation of a single
were synthesised using phosphoramidite dimers 1 and 2, which triazole linkage (ON10) decreased the thermal stability of the
were fully compatible with the sulfurisation step used during duplex by 4.3 1C, triple addition (ON11) reduced the Tm by
solid-phase ON synthesis. The ONs were then labelled with PY- 14.7 1C and quadruple addition (ON12) dropped the Tm by

768 | RSC Chem. Biol., 2022, 3, 765–772 © 2022 The Author(s). Published by the Royal Society of Chemistry
 View Article Online

RSC Chemical Biology Paper

20.8 1C. These results might suggest that the triazole is better
tolerated in an A-form PS/2 0 -OMe backbone than a B-form DNA
backbone (compare ON1 : RNA DTm = 7.4 1C with ON10 : RNA
DTm = 4.3); however, as these data were recorded in different
sequences caution must be applied when drawing compari-
sons. As before, addition of PY and AQ to the triazole modified
ONs restored some of the lost stability. ON13 with a single
triazole linkage and PY showed a Tm increase of 5.2 1C com-
pared to the triazole alone in ON10 and exceeded the Tm of the
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

PS/2 0 -OMe control ON19 without a triazole by 0.9 1C. Similarly,

addition of AQ to ON10 increased the stability by 8.2 1C
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

(Compare ON10 with ON16), which is 3.9 1C higher than the

unmodified control ON19. Fig. 5 Splice-switching activity of transfected antisense oligonucleotides
The stabilising effect of AQ and PY with triple and quadruple in the stably transfected HeLa Luc/705 cell line (assay 1) using Lipofecta-
mine 2000. Cells were treated for 24 h. Experiments were replicated with
additions of the triazole linkage was not as great as anticipated. data showing standard deviation of the mean (SD).
Adding PY to ON11 with three triazole linkages only increased
the Tm by 5.0 1C (ON14), and the resulting duplex was 9.7 1C
less stable than the control (ON19). ON15 with four PY functio- most to least active oligonucleotides are ON13 (Tm = 71.1 1C) 4
nalised triazole linkages was only 7.3 1C more stable than ON12 ON10 (Tm = 65.9 1C) 4 ON19 (Tm = 70.2 1C) 4 ON14 (Tm =
without PY, and 13.5 1C less stable than the control. More 60.5 1C) 4 ON15 (Tm = 56.7 1C) 4 ON11 (Tm = 55.5 1C) 4 ON12
surprisingly, addition of three and four anthraquinone moi- (Tm = 49.4 1C) suggesting that duplex stability correlates with
eties next to triazole linkages (ON17 Tm = 51.1 1C and ON18 splice switching efficacy. It is clear that the addition of pyrene
Tm = 45.1 1C) caused a decrease in duplex stability compared to to triazole modified ONs improves their splice switching activ-
the triazole containing ON11 (Tm = 55.5 1C) and ON12 ity; a higher response is observed when comparing ON13 with
(Tm = 49.4 1C). ON10 at the lower concentrations, and ON14 and ON15 with PY
modifications are significantly more active than their triazole-
Biological evaluation of the pyrene triazole combination only equivalents ON11 and ON12.
Having confirmed that pyrene is more capable than anthraqui- In order to substantiate these findings, a second luciferase
none of compensating for the loss of duplex stability incurred exon skipping assay was performed, targeting the C to T
with multiple triazoles in PS/2 0 -OMe ASOs, we evaluated efficacy nucleotide mutation at position 654 in human b-globin intron
of the modified ASOs in a commonly used splice-switching 2, a mutation that also causes b-thalassemia. In this assay,
reporter cell system.48 This model system is based on a muta- MCF7 cells are transduced with a pHTBV1 plasmid encoding
tion that causes the blood disorder b-thalassemia. The HeLa luciferase, interrupted with IVS2-695, using BacMam one day
pLuc705 cells used had been stably transfected with a plasmid prior to treatment with the ONs. The sequences of the ONs used
that encodes for luciferase interrupted by a mutated human b- and their duplex melting temperatures are shown in Table 3. As
globin intron 2 (IVS2-705). This single point mutation causes before, PY restored the duplex stability lost from the triazole
aberrant splicing, preventing translation of active luciferase. incorporation, although some sequence dependence was
Masking the aberrant splice site with an ASO redirects the observed. Lipofection with ON20-24 in a concentration range
splicing machinery and restores luciferase production. ONs of 91 pM to 22.2 nM resulted in a dose dependent response for
used in the Tm studies (Table 2) were designed to be comple- all ONs tested (Fig. 6, top graph) and toxicity was observed at
mentary to the splice site, and ON19 is the standard control higher concentrations. Again, the data show that the triazole
sequence used with this assay.
The splice switching assay confirmed that replacement of
the PS backbone with more than one triazole is detrimental to Table 3 Sequences and thermal melting data for the PS/2 0 -OMe ONs
used in assay. Melting temperatures against complementary RNA
activity and showed that the addition of pyrene to the triazole
modified ASO re-establishes splice switching. The addition of ON ON sequence (5 0 to 3 0 ) Tm 1C
PY also improved the response of the ASO with a single triazole
20 72.8
relative to the parent ASO (Fig. 5). Here, the cells were treated
21 60.5
with ON10-15 complexed with Lipofectamine 2000 in the
22 68.1
concentration range 7.8 nM to 180 nM. At the higher concen-
23 66.1
tration a maximal effect is observed for ON10, ON13, ON15 and
24 70.7
ON19 (around 40-fold increase in activity). Higher doses of
oligonucleotide-lipofectamine complexes caused significant X = AE–T; = pyrene labelled AE–T; = position of a triazole in place
cell toxicity. A clear dose response was observed for all oligo- of the phosphorothioate; = triazole dimer 1. Melting temperatures
were determined at 2 mM duplex concentration in 10 mM NaH2PO4/
nucleotides except for ON12 with four triazoles and no PY, Na2HPO4 buffer with 200 mM NaCl at pH 7.2. Sequence of RNA target =
which was inactive. These dose responses indicate that the CUGGGUUAAGGUAAUAGC.

© 2022 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2022, 3, 765–772 | 769
 View Article Online

Paper RSC Chemical Biology

provided a convenient point for post-synthetic functionalisa-

tion with electrophilic reagents. The effects on duplex stability
of cationic and intercalating agents positioned adjacent to the
triazole linkage were then compared. Having identified that
addition of the AQ or PY modifications restores the stability lost
by inclusion of the triazole, we evaluated these linkages in exon-
skipping ASOs using two splice switching assays. As antici-
pated, the triazole alone resulted in lower activity; however,
addition of PY to the ONs fully or partially restored the lost
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

activity and, in some cases, the combination resulted in more

potent ASO than the parent ASO with a standard 2 0 -OMe-
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

phosphorothioate backbone. Taken together, these results

indicate that the triazole linkage combined with PY or AQ
could be a useful addition to the ASO chemical toolkit and is
worth further exploration. Placing the modifications in the gap
or the wings of antisense oligonucleotides, is also worth
investigating to determine if they interfere with the activity of
RNase-H. Use of the modifications in siRNA might also feasible.
In general, future research, including optimisation of the
linkage between the nucleoside and the PY or AQ, might
produce oligonucleotides with increased potency.
The approach used here can also potentially be applied to
other backbone modifications which may improve an ASO’s
pharmacokinetic properties but have previously taken a back-
seat due to duplex stability challenges. It also provides a route
for the rapid screening of chemical attachments, beyond the
three functionalities presented here, and could be used to
Fig. 6 Splice-switching activity of triazole-modified ASOs in MCF7 cells
transduced with a pHTBV1 plasmid encoding luciferase interrupted with evaluate a plethora of moieties for endosomal escape and/or
IVS2-695 (assay 2). (Top) Cells were treated for 24 h with lipofectamine : enhanced duplex stability.
ASO complexes. (Bottom) Gymnotic delivery experiments. Cells were
treated with the ASO for 24 without a transfection agent. Experiments
were performed in triplicate with data showing standard deviation (SD). Conflicts of interest
There are no conflicts to declare.
alone reduces the efficacy of the ASO and that adding a PY
restores the activity of the ASO. As before, a correlation between
the Tm and activity was observed, with the exception of ON24 Acknowledgements
(Tm = 70.7 1C) appearing slightly more active that ON20 (Tm =
This work was supported by UK BBSRC grants BB/J001694/2
72.8 1C).
(Extending the boundaries of nucleic acid chemistry), BB/
The effect of modifications on gymnotic (naked) delivery was
R008655/1 (New and versatile chemical approaches for the
also determined using this assay (Fig. 6 bottom graph). As with
synthesis of mRNA and tRNA) and BB/S018794/1 (New oligo-
the lipofection experiments PY improved the activity of the
nucleotide analogues for therapeutic applications). The authors
triazole modified ONs. Double addition of the triazole without
would like to thank Dr Samir El-Andaloussi for the HeLa pLuc/
PY (ON21) also had a less detrimental effect on activity when
705 cell line and Dr Susan Merrihew for valuable discussions.
compared to the parent ASO (ON20) than was observed with
This research utilised equipment funded by the John Fell
lipofection. This could be a result of increased lipophilicity
Oxford University Press Research Fund and an EPSRC Strategic
aiding endosomal escape or cell uptake. Further investigations
Equipment Grant (EP/T019190/1).
are required to clarify this.

References
Conclusions
1 C. I.-E. Smith and R. Zain, Therapeutic Oligonucleotides:
We have prepared two novel dinucleotide phosphoramidite State of the Art, Annu. Rev. Pharmacol. Toxicol., 2019, 59,
monomers containing a triazole internucleoside linkage and 605–630.
a 2 0 -aminoethoxy modification on the 5 0 -side of the triazole. 2 A. Khvorova and J. K. Watts, The chemical evolution of
The building blocks were readily incorporated into oligonucleo- oligonucleotide therapies of clinical utility, Nat. Biotechnol.,
tides during solid phase oligonucleotide synthesis and 2017, 35, 238–248.

770 | RSC Chem. Biol., 2022, 3, 765–772 © 2022 The Author(s). Published by the Royal Society of Chemistry
 View Article Online

RSC Chemical Biology Paper

3 S. T. Crooke, X.-H. Liang, B. F. Baker and R. M. Crooke, 20 A. H. El-Sagheer, A. P. Sanzone, R. Gao, A. Tavassoli and
Antisense technology: A review, J. Biol. Chem., 2021, T. Brown, Biocompatible artificial DNA linker that is read
296, 100416. through by DNA polymerases and is functional in Escher-
4 S. T. Crooke, Molecular Mechanisms of Antisense Oligonu- ichia coli, Proc. Natl. Acad. Sci. U. S. A., 2011, 108,
cleotides, Nucleic Acid Ther., 2017, 27, 70–77. 11338–11343.
5 X.-H. Liang, H. Sun, J. G. Nichols and S. T. Crooke, RNase 21 P. Kumar, A. H. El-Sagheer, L. Truong and T. Brown, Locked
H1-Dependent Antisense Oligonucleotides Are Robustly nucleic acid (LNA) enhances binding affinity of triazole-
Active in Directing RNA Cleavage in Both the Cytoplasm linked DNA towards RNA, Chem. Commun., 2017, 53,
and the Nucleus, Mol. Ther., 2017, 25, 2075–2092. 8910–8913.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

6 C. Rinaldi and M. J. A. Wood, Antisense oligonucleotides: 22 Y. R. Baker, et al., Searching for the ideal triazole: Investi-
the next frontier for treatment of neurological disorders, gating the 1,5-triazole as a charge neutral DNA backbone
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

Nat. Rev. Neurol., 2018, 14, 9–21. mimic, Tetrahedron, 2020, 76, 130914.
7 Z. Dominski and R. Kole, Restoration of correct splicing in 23 A. H. El-Sagheer and T. Brown, Combined nucleobase and
thalassemic pre-mRNA by antisense oligonucleotides, Proc. backbone modifications enhance DNA duplex stability and
Natl. Acad. Sci. U. S. A., 1993, 90, 8673–8677. preserve biocompatibility, Chem. Sci., 2014, 5, 253–259.
8 H. Sierakowska, M. J. Sambade, S. Agrawal and R. Kole, 24 M. J. Palframan, R. D. Alharthy, P. K. Powalowska and
Repair of thalassemic human beta-globin mRNA in mam- C. J. Hayes, Synthesis of triazole-linked morpholino oligo-
malian cells by antisense oligonucleotides, Proc. Natl. Acad. nucleotides via Cu(I) catalysed cycloaddition, Org. Biomol.
Sci. U. S. A., 1996, 93, 12840–12844. Chem., 2016, 14, 3112–3119.
9 A. Aartsma-Rus, et al., Therapeutic antisense-induced exon 25 P. Kumar, L. Truong, Y. R. Baker, A. H. El-Sagheer and
skipping in cultured muscle cells from six different DMD T. Brown, Synthesis, Affinity for Complementary RNA and
patients, Hum. Mol. Genet., 2003, 12, 907–914. DNA, and Enzymatic Stability of Triazole-Linked Locked
10 L. K. McKenzie, R. El-Khoury, J. D. Thorpe, M. J. Damha and Nucleic Acids (t-LNAs), ACS Omega, 2018, 3, 6976–6987.
M. Hollenstein, Recent progress in non-native nucleic acid 26 V. K. Sharma, et al., Synthesis and biological properties of
modifications, Chem. Soc. Rev., 2021, 50, 5126–5164. triazole-linked locked nucleic acid, Chem. Commun., 2017,
11 B. P. Monia, J. F. Johnston, H. Sasmor and L. L. Cummins, 53, 8906–8909.
Nuclease resistance and antisense activity of modified oli- 27 A. M. Varizhuk, et al., Synthesis of triazole-linked oligonu-
gonucleotides targeted to Ha-ras, J. Biol. Chem., 1996, 271, cleotides with high affinity to DNA complements and an
14533–14540. analysis of their compatibility with biosystems, J. Org.
12 F. Eckstein, Phosphorothioates, essential components of Chem., 2013, 78, 5964–5969.
therapeutic oligonucleotides, Nucleic Acid Ther., 2014, 24, 28 H. Isobe, T. Fujino, N. Yamazaki, M. Guillot-Nieckowski and
374–387. E. Nakamura, Triazole-linked analogue of deoxyribonucleic
13 L. L. Cummins, et al., Characterization of fully 2’-modified acid ((TL)DNA): design, synthesis, and double-strand for-
oligoribonucleotide hetero- and homoduplex hybridization mation with natural DNA, Org. Lett., 2008, 10, 3729–3732.
and nuclease sensitivity, Nucleic Acids Res., 1995, 23, 29 R. Lucas, et al., Microwave-assisted synthesis of a triazole-
2019–2024. linked 3 ’-5 ’ dithymidine using click chemistry, Tetrahedron
14 P. Martin, A New Access to 2 0 -O-Alkylated Ribonucleosides Lett., 2008, 49, 1004–1007.
and Properties of 2 0 -O-Alkylated Oligoribonucleotides, Helv. 30 K. Yamana, et al., 2 0 -Pyrene modified oligonucleotide pro-
Chim. Acta, 1995, 78, 486–504. vides a highly sensitive fluorescent probe of RNA, Nucleic
15 A. A. Koshkin, et al., LNA (Locked Nucleic Acids): Synthesis Acids Res., 1999, 27, 2387–2392.
of the adenine, cytosine, guanine, 5-methylcytosine, thy- 31 P. Grünefeld and C. Richert, Synthesis of a 1‘-Amino-
mine and uracil bicyclonucleoside monomers, oligomerisa- methylthymidine and Oligodeoxyribonucleotides with 1‘-
tion, and unprecedented nucleic acid recognition, Acylamidomethylthymidine Residues, J. Org. Chem., 2004, 69,
Tetrahedron, 1998, 54, 3607–3630. 7543–7551.
16 F. Wang, T. Zuroske and J. K. Watts, RNA therapeutics on 32 M. E. Ostergaard and P. J. Hrdlicka, Pyrene-functionalized
the rise, Nat. Rev. Drug. Discov., 2020, 19, 441–442. oligonucleotides and locked nucleic acids (LNAs): Tools for
17 D. Sheehan, et al., Biochemical properties of phosphonoa- fundamental research, diagnostics, and nanotechnology,
cetate and thiophosphonoacetate oligodeoxyribonucleo- Chem. Soc. Rev., 2011, 40, 5771–5788.
tides, Nucleic Acids Res., 2003, 31, 4109–4118. 33 M. Nakamura, et al., Pyrene is highly emissive when
18 B. R. Meade, et al., Efficient delivery of RNAi prodrugs contain- attached to the RNA duplex but not to the DNA duplex:
ing reversible charge-neutralizing phosphotriester backbone the structural basis of this difference, Nucleic Acids Res.,
modifications, Nat. Biotechnol., 2014, 32, 1256–1261. 2005, 33, 5887–5895.
19 Y. Shoji, S. Akhtar, A. Periasamy, B. Herman and 34 N. Ben Gaied, Z. Zhao, S. R. Gerrard, K. R. Fox and T. Brown,
R. L. Juliano, Mechanism of cellular uptake of modified Potent Triple Helix Stabilization by 5 ’,3 ’-Modified Triplex-
oligodeoxynucleotides containing methylphosphonate lin- Forming Oligonucleotides, ChemBioChem, 2009, 10,
kages, Nucleic Acids Res., 1991, 19, 5543–5550. 1839–1851.

© 2022 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2022, 3, 765–772 | 771
 View Article Online

Paper RSC Chemical Biology

35 Z. Zhao, G. Peng, J. Michels, K. R. Fox and T. Brown, 42 W. Bannwarth, Solid-Phase Synthesis of Oligodeoxynucleo-
Synthesis of anthraquinone oligonucleotides for triplex tides containing phosphoramidate internucleotide linkages
stabilization, Nucleosides, Nucleotides Nucleic Acids, 2007, and their specific chemical cleavage, Helv. Chim. Acta, 1988,
26, 921–925. 71, 1517–1527.
36 M. B. Gholivand, S. Kashanian and H. Peyman, DNA- 43 I. Yamamoto, M. Sekine and T. Hata, One-step synthesis os
binding, DNA cleavage and cytotoxicity studies of two 5’-azido-nucleosides, J. Chem. Soc., Perkin Trans. 1, 1980,
anthraquinone derivatives, Spectrochim. Acta, Part A, 2012, 306–310.
87, 232–240. 44 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, A
37 K. Mori, C. Subasinghe and J. S. Cohen, Oligodeoxynucleo- Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

tide analogs with 5 0 -linked anthraquinone, FEBS Lett., 1989, Regioselective ‘‘Ligation’’ of Azides and Terminal Alkynes,
249, 213–218. Angew. Chem., Int. Ed., 2002, 41, 2596–2599.
Open Access Article. Published on 16 May 2022. Downloaded on 5/16/2025 [Link] AM.

38 G. Deglane, et al., Impact of the guanidinium group on 45 K. S. Chamberlin, Acid-aided selectivity in bromination of 1-
hybridization and cellular uptake of cationic oligonucleo- aminoathraquinones, Synth. Commun., 1995, 25, 27–31.
tides, ChemBioChem, 2006, 7, 684–692. 46 R. Ohrlein and G. Baisch, in US pat. 2009/0203931A11-26,
39 T. P. Prakash, et al., 2 0 -O-2-(Guanidinium)ethyl-modified Ciba Corporation, USA, 2009.
oligonucleotides: Stabilizing effect on duplex and triplex 47 S. M. Freier and K.-H. Altmann, The ups and downs of
structures, Org. Lett., 2004, 6, 1971–1974. nucleic acid duplex stability: Structure-stability studies on
40 K. Ohara, et al., Amine-guanidine switch: A promising chemically-modified DNA : RNA duplexes, Nucleic Acids Res.,
approach to improve DNA binding and antiproliferative 1997, 25, 4429–4443.
activities, J. Med. Chem., 2007, 50, 6465–6475. 48 S.-H. Kang, M.-J. Cho and R. Kole, Up-Regulation of Lucifer-
41 J. A. Richardson, M. Gerowska, M. Shelbourne, D. French ase Gene Expression with Antisense Oligonucleotides:
and T. Brown, Six-Colour HyBeacon Probes for Multiplex Implications and Applications in Functional Assay Develop-
Genetic Analysis, ChemBioChem, 2010, 11, 2530–2533. ment, Biochemistry, 1998, 37, 6235–6239.

772 | RSC Chem. Biol., 2022, 3, 765–772 © 2022 The Author(s). Published by the Royal Society of Chemistry